Project Summary Alzheimer's disease (AD) is the most common neurodegenerative disorder and a leading cause of disability and death. However, the precise mechanisms underlying AD pathogenesis remains to be elucidated. Although many transgenic mouse models have been generated for AD research and these models are important for our understanding of the pathological basis of the disease, none has captured the entire spectrum of the disease pathology, including considerable neuronal loss. This is likely due to significant species differences between mouse and human neural cells. Therefore, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for AD research and drug development. Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, human iPSCs (hiPSCs) have been widely used for disease modeling and drug discovery. However, given the relative immaturity of cells differentiated from hiPSC, it is challenging to use them to model late-onset diseases, for which cellular aging is important in disease pathology. Direct reprogramming is an alternative cellular reprogramming technology, which allows direct conversion of one type of somatic cells, such as fibroblasts, into another type of somatic cells, such as neurons. It has been shown that direct reprogramming enables generation of human neurons that possess key elements of cellular aging, because this reprogramming process does not go through the rejuvenating iPSC stage. The objective of this proposal is to develop aging-relevant cellular models of late-onset AD (LOAD), using direct reprogramming technology in combination with CRISPR/Cas9-mediated gene editing and 3D neural culture, in order to recapitulate the age-associated phenotypes and uncover novel pathological mechanisms of LOAD. We propose to establish cellular models of LOAD using both neurons and astrocytes directly reprogrammed from patient fibroblasts or differentiated from induced neural stem cells (iNSCs) obtained through direct reprogramming. While the strongest risk factor for AD is aging, the strongest genetic risk factor of AD is apolipoprotein (apo) E4. We hypothesize that cellular aging and apoE genotype interact with each other to initiate and/or modulate LOAD pathologies. Accordingly, we propose three complementary aims to test this hypothesis. Aim 1: To generate isogenic human fibroblast lines with different apoE genotypes as cell sources for direct reprogramming. Aim 2: To model LOAD using directly reprogrammed human neurons and astrocytes. Aim 3: To model LOAD using directly reprogrammed NSC-derived neurons and astrocytes. The outcomes of the proposed studies will likely help to further define the roles of apoE4 in the development of age-associated AD pathological features, to uncover novel mechanisms for age and apoE4-related AD pathogenesis, and to design novel therapeutic strategies for AD.